Biology Reference
In-Depth Information
CHAPTER
4
Geoff Doherty
1
, Karla Mettrick
1
, Ian Grainge, Peter J. Lewis
2
School of Environmental and Life Sciences, University of Newcastle, Callaghan,
New South Wales, Australia
2
Corresponding author. e-mail address: Peter.Lewis@newcastle.edu.au
Imaging fluorescent protein
fusions in live bacteria
1
INTRODUCTION
Although they lack the defined subcellular organelles that eukaryotes use to partition
cellular processes, it has become evident over the past 15 years that distinct subcel-
lular domains are present in prokaryotes. The majority of our knowledge of subcel-
lular compartmentalisation has arisen through the use of fluorescent tags such as GFP
and its derivatives. The approaches utilised to identify protein localisation and abun-
dance are suitable for high-throughput systems biology projects (
Meile
et al.
, 2006;
Werner
et al.
, 2009; Buescher
et al.
, 2012
). Here, we describe some of the molecular
toolkits currently available to observe the subcellular localisation of both fully and
partially functional fusions in a variety of prokaryotes, focussing on
Bacillus subtilis
,
Escherichia coli
,
Staphylococcus aureus
and
Acinetobacter baylyi
.
2
MOLECULAR TOOLKITS
There are a variety of tools available for labelling proteins in live, intact cells. Most
of the tools comprise gene fusions constructed on a plasmid that can be either inte-
grative or non-integrative, which are then transformed into the target cell. Alterna-
tively, simple PCR constructs can be used to create gene fusions relying upon
homologous recombination for integration. With an explosion of fluorescent tags
having entered the cytological ball park since Martin Chalfie first described the
use of GFP from jellyfish
Aequorea victoria
(
Chalfie
et al.
, 1994
), researchers have
a myriad of choices when undertaking localisation studies. These include GFP var-
iants that have improved biophysical properties and shifted spectra (
Chalfie
et al.
,
1994; Heim
et al.
, 1995; Cormack
et al.
, 1996; Heim and Tsien, 1996
), and
also include the more recently developed mFruits, which are monomeric variants
of the
Discosoma
genus of coral fluorescent protein dsRed (
Shu
et al.
, 2006
).
1
Contributed equally to this work.
